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Biomedical subjects

Mark S Kindy

Publications and source records attributed to Mark S Kindy.

17 recordsLinked to original sources
PubMed2007

Estradiol enhances neurogenesis following ischemic stroke through estrogen receptors alpha and beta.

Neurogenesis persists throughout life under normal and degenerative conditions. The adult subventricular zone (SVZ) generates neural stem cells capable of differentiating to neuroblasts and migrating to the site of injury in response to brain insults. In the present study, we investigated whether estradiol increases neurogenesis in the SVZ in an animal model of stroke to potentially promote the ability of the brain to undergo repair. Ovariectomized C57BL/6J mice were implanted with capsules containing either vehicle or 17beta-estradiol, and 1 week later they underwent experimental ischemia. We utilized double-label immunocytochemistry to identify the phenotype of newborn cells (5-bromo-2'-deoxyuridine-labeled) with various cellular markers; doublecortin and PSA-NCAM as the early neuronal marker, NeuN to identify mature neurons, and glial fibrillary acidic protein to identify astrocytes. We report that low physiological levels of estradiol treatment, which exert no effect in the uninjured state, significantly increase the number of newborn neurons in the SVZ following stroke injury. This effect of estradiol is limited to the dorsal region of the SVZ and is absent from the ventral SVZ. The proliferative actions of estradiol are confined to neuronal precursors and do not influence gliosis. Furthermore, we show that both estrogen receptors alpha and beta play pivotal functional roles, insofar as knocking out either of these receptors blocks the ability of estradiol to increase neurogenesis. These findings clearly demonstrate that estradiol stimulates neurogenesis in the adult SVZ, thus potentially facilitating the brain to remodel and repair after injury.

Animals↗
PubMed2006

Complement-dependent P-selectin expression and injury following ischemic stroke.

The mechanisms that contribute to inflammatory damage following ischemic stroke are poorly characterized, but studies indicate a role for both complement and P-selectin. In this study, we show that compared with wild-type mice, C3-deficient mice showed significant improvement in survival, neurological deficit, and infarct size at 24 h after middle cerebral artery occlusion and reperfusion. Furthermore, P-selectin protein expression was undetectable in the cerebral microvasculature of C3-deficient mice following reperfusion, and there was reduced neutrophil influx, reduced microthrombus formation, and increased blood flow postreperfusion in C3-deficient mice. We further investigated the use of a novel complement inhibitory protein in a therapeutic paradigm. Complement receptor 2 (CR2)-Crry inhibits complement activation at the C3 stage and targets to sites of complement activation. Treatment of normal mice with CR2-Crry at 30 min postreperfusion resulted in a similar level of protection to that seen in C3-deficient mice in all of the above-measured parameters. The data demonstrate an important role for complement in cerebrovascular thrombosis, inflammation, and injury following ischemic stroke. P-selectin expression in the cerebrovasculature, which is also implicated in cerebral ischemia and reperfusion injury, was shown to be distal to and dependent on complement activation. Data also show that a CR2-targeted approach of complement inhibition provides appropriate bioavailability in cerebral injury to enable complement inhibition at a dose that does not significantly affect systemic levels of serum complement activity, a potential benefit for stroke patients where immunosuppression would be undesirable due to significantly increased susceptibility to lung infection.

Animals↗
PubMed2006

A dolphin peripheral blood leukocyte cDNA microarray for studies of immune function and stress reactions.

A microarray focused on stress response and immune function genes of the bottlenosed dolphin has been developed. Random expressed sequence tags (ESTs) were isolated and sequenced from two dolphin peripheral blood leukocyte (PBL) cDNA libraries biased towards T- and B-cell gene expression by stimulation with IL-2 and LPS, respectively. A total of 2784 clones were sequenced and contig analysis yielded 1343 unigenes (archived and annotated at ). In addition, 52 dolphin genes known to be important in innate and adaptive immune function and stress responses of terrestrial mammals were specifically targeted, cloned and added to the unigene collection. The set of dolphin sequences printed on a cDNA microarray comprised the 1343 unigenes, the 52 targeted genes and 2305 randomly selected (but unsequenced) EST clones. This set was printed in duplicate spots, side by side, and in two replicates per slide, such that the total number of features per microarray slide was 19,200, including controls. The dolphin arrays were validated and transcriptomic profiles were generated using PBL from a wild dolphin, a captive dolphin and dolphin skin cells. The results demonstrate that the array is a reproducible and informative tool for assessing differential gene expression in dolphin PBL and in other tissues.

Animals↗
PubMed2006

Differential modulation of estrogen receptors (ERs) in ischemic brain injury: a role for ERalpha in estradiol-mediated protection against delayed cell death.

Estradiol enhances plasticity and survival of the injured brain. Our previous work demonstrates that physiological levels of estradiol protect against cerebral ischemia in the young and aging brain through actions involving estrogen receptors (ERs) and alterations in gene expression. The major goal of this study was to establish mechanisms of neuroprotective actions induced by low levels of estradiol. We first examined effects of estradiol on the time-dependent evolution of ischemic brain injury. Because estradiol is known to influence apoptosis, we hypothesized that it acts to decrease the delayed phase of cell death observed after middle cerebral artery occlusion (MCAO). Furthermore, because ERs are pivotal to neuroprotection, we examined the temporal expression profiles of both ER subtypes, ERalpha and ERbeta, after MCAO and delineated potential roles for each receptor in estradiol-mediated neuroprotection. We quantified cell death in brains at various times after MCAO and analyzed ER expression by RT-PCR, in situ hybridization, and immunohistochemistry. We found that during the first 24 h, the mechanisms of estradiol-induced neuroprotection after MCAO are limited to attenuation of delayed cell death and do not influence immediate cell death. Furthermore, we discovered that ERs exhibit distinctly divergent profiles of expression over the evolution of injury, with ERalpha induction occurring early and ERbeta modulation occurring later. Finally, we provide evidence for a new and functional role for ERalpha in estradiol-mediated protection of the injured brain. These findings indicate that physiological levels of estradiol protect against delayed cell death after stroke-like injury through mechanisms requiring ERalpha.

Animals↗
PubMed2005

Establishment of epidermal cell lines derived from the skin of the Atlantic bottlenose dolphin (Tursiops truncatus).

The Atlantic bottlenose dolphin (Tursiops truncatus), a marine mammal found off the Atlantic coast, has become the focus of considerable attention because of an increasing number of mortality events witnessed in this species over the last several years along the southeastern United States. Assessment of the impact of environmental stressors on bottlenose dolphins (BND) has been difficult because of the protected status of these marine mammals. The studies presented herein focused on establishing epidermal cell cultures and cell lines as tools for the in vitro evaluation of environmental stressors on BND skin. Epidermal cell cultures were established from skin samples obtained from Atlantic BND and subjected to karyotype analysis. These cultures were further characterized using immunohistochemical methods demonstrating expression of cytokeratins. By two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we observed that the proteomic profile of BND skin tissue samples shared distinct similarities with that of skin-derived cultures. Epidermal cell cultures were transfected with a plasmid encoding the SV40 small t- and large T-antigens, as well as the neomycin-resistance gene. Five neomycin-resistant clones were isolated and expanded, and all of them proliferated at a faster rate than nontransfected BND epidermal cultures, which exhibited signs of senescence. Cell lysates prepared from two transfected clones were shown to express, by Western blot analysis, both SV40 tumor antigens. These experimental results are consistent with the concept that transfected clones expressing SV40 tumor antigens represent immortalized BND cell lines. Epidermal cell lines derived from Tursiops truncatus will provide a unique tool for studying key features of the interaction occurring between dolphins and the environment in which they live at their most crucial interface: the skin.

Animals↗
PubMed2004

Activation of nuclear factor kappaB In vivo selectively protects the murine small intestine against ionizing radiation-induced damage.

Exposure of mice to total body irradiation induces nuclear factor kappaB (NFkappaB) activation in a tissue-specific manner. In addition to the spleen, lymph nodes, and bone marrow, the tissues that exhibit NFkappaB activation now include the newly identified site of the intestinal epithelial cells. NFkappaB activated by total body irradiation mainly consists of NFkappaB p50/RelA heterodimers, and genetically targeted disruption of the NFkappaB p50 gene in mice significantly decreased the activation. By comparing tissue damage and lethality in wild-type and NFkappaB p50 knockout (p50-/-) mice after they were exposed to increasing doses of total body irradiation, we additionally examined the role of NFkappaB activation in total body irradiation-induced tissue damage. The results show that p50-/- mice are more sensitive to total body irradiation-induced lethality than wild-type mice (LD50/Day 7: wild-type = 13.12 Gy versus p50-/- = 7.75 Gy and LD50/Day 30: wild-type = 9.31 Gy versus p50-/- = 7.81 Gy). The increased radiosensitivity of p50-/- mice was associated with an elevated level of apoptosis in intestinal epithelial cells and decreased survival of the small intestinal crypts compared with wild-type mice (P < 0.01). In addition, RelA/TNFR1-deficient (RelA/TNFR1-/-) mice also exhibited a significant increase in intestinal epithelial cell apoptosis after they were exposed to total body irradiation as compared with TNFR1-deficient (TNFR1-/-) mice (P < 0.01). In contrast, no significant increase in total body irradiation-induced apoptosis or tissue injury was observed in bone marrow cells, spleen lymphocytes, and the liver, heart, lung, and kidney of p50-/- mice in comparison with wild-type mice. These findings indicate that activation of NFkappaB selectively protects the small intestine against ionizing radiation-induced damage.

Animals↗
PubMed2004

Time window of fibroblast growth factor-18-mediated neuroprotection after occlusion of the middle cerebral artery in rats.

To assess the time window for fibroblast growth factor-18 (FGF18)-mediated neuroprotection, FGF18 was administered by intravenous infusion at various times after transient occlusion of the middle cerebral artery (MCAO) in rats. Vehicle or FGF18 (100 microg x kg(-1) x h(-1)) was infused at 0.25, 0.5, 1.0, 2.0, 4.0, or 8.0 hours after MCAO with infarct volumes and behavioral deficits measured at 24.0 hours after MCAO. A separate group of animals received the infusions 24 hours after MCAO with endpoints measured at 48 hours after MCAO. Infusion of FGF18 reduced infarct volumes and improved scores in tests of reference and working memory, motor ability, and exploratory behavior. FGF18 was most efficacious when infused within 2 hours after MCAO. Significant reductions in infarct volumes and reductions in deficits of reference memory and motor activity were also observed with FGF18 infused 24 hours after MCAO. Measurements taken at infusion times before 2 hours after MCAO showed that regional cerebral blood flow was increased by FGF18. Administration of vehicle or FGF18 had no significant effect on mean arterial blood pressure, heart rate, brain temperature, blood pH, Pco2, or Po2. These results demonstrate that FGF18 is an effective neuroprotective agent when administered early after transient MCAO in rats. Efficacy observed with infusions at later times suggests an expanded time window for FGF18-mediated neuroprotection.

Animals↗
PubMed2004

Cholesterol and Alzheimer's disease: clinical and experimental models suggest interactions of different genetic, dietary and environmental risk factors.

Alzheimer's disease (AD) is a progressive senile dementia characterized by deposition of a 4 kDa peptide of 39-42 residues known as amyloid beta-peptide (Abeta) in the form of senile plaques and the microtubule associated protein tau as paired helical filaments. Genetic studies have identified mutations in the Abeta precursor protein (APP) as the key triggers for the pathogenesis of AD. Other genes such as presenilins 1 and 2 (PS1/2) and apolipoprotein E (APOE) also play a critical role in increased Abeta deposition. Several biochemical and molecular studies using transfected cultured cells and transgenic animals point to mechanisms by which Abeta is generated and aggregated to trigger the neurodegeneration that may cause AD. Three important enzymes collectively known as 'secretases' participate in APP processing leading to the generation of either Abeta or non-amyloid proteins. However, the mechanisms of neurotoxicity of Abeta and the role of APP function in AD remain important unanswered questions. Although early studies recognized the loss of cholesterol and other lipids in the brain, these findings have been poorly connected with AD pathogenesis, despite the identification of the epsilon4 allele of APOE as a major risk factor in AD. The recent finding that cholesterol can modulate the yield of potentially toxic Abeta has boosted research on its role in AD. Consequently, several cholesterol-reducing drugs are currently being evaluated for the treatment of AD. The present review summarizes our current understanding of the relationship of AD pathogenesis with cholesterol, lipids and other genetic and environmental risk factors.

Alzheimer Disease↗
PubMed2003

In vitro and in vivo modulations of benzo[c]phenanthrene-DNA adducts by DNA mismatch repair system.

Benzo[c]phenanthrene dihydrodiol epoxide (B[c] PhDE) is well known as an important environmental chemical carcinogen that preferentially modifies DNA in adenine residues. However, the molecular mechanism by which B[c]PhDE induces tumorigenesis is not fully understood. In this report, we demonstrate that DNA mismatch repair (MMR), a genome maintenance system, plays an important role in B[c]PhDE-induced carcinogensis by promoting apoptosis in cells treated with B[c]PhDE. We show that purified human MMR recognition proteins, MutS(alpha) and MutSbeta, specifically recognized B[c]PhDE-DNA adducts. Cell lines proficient in MMR exhibited several-fold more sensitivity to killing than cell lines defective in either MutS(alpha) or MutL(alpha) by B[c]PhDE; the nature of this sensitivity was shown to be due to increased apoptosis. Additionally, wild-type mice exposed to B[c]PhDE had intestinal crypt cells that underwent apoptosis significantly more often than intestinal crypt cells found in B[c]PhDE-treated Msh2(-/-) or Mlh1(-/-) mice. These findings, combined with previous studies, suggest that the MMR system may serve as a general sensor for chemical-caused DNA damage to prevent damaged cells from mutagenesis and carcinogenesis by promoting apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Fibroblast growth factor-18 reduced infarct volumes and behavioral deficits after transient occlusion of the middle cerebral artery in rats.

BACKGROUND AND PURPOSE: Fibroblast growth factor 18 (FGF18) is expressed in rodent brain and is a trophic factor for neuron-derived cells in culture. The purpose of the present study was to evaluate whether FGF18 was neuroprotective in a rat model of cerebral ischemia and to compare the results with those obtained with FGF2. METHODS: Cerebral ischemia was produced in rats by a transient 2-hour occlusion of the middle cerebral artery (MCAo) with an intraluminal filament followed by 22-hour reperfusion. Starting 15 minutes after MCAo, FGF18 or FGF2 was administered by a 3-hour intravenous infusion. Infarct volumes and behavioral deficits were measured 24 hours after MCAo. RESULTS: Infusion of FGF18 produced dose-dependent reductions in infarct volumes and improvements in tests of reference and working memory, motor ability, and exploratory behavior. FGF18 was more efficacious than FGF2 on virtually all measures examined. The reductions in infarct volume and behavioral deficit were associated with FGF-mediated increases in regional cerebral blood flow. CONCLUSIONS: These results demonstrate that FGF18 is an effective neuroprotective agent in a rat model of transient MCAo.

Animals↗
PubMed2003

Neprilysin gene transfer reduces human amyloid pathology in transgenic mice.

The degenerative process of Alzheimer's disease is linked to a shift in the balance between amyloid-beta (Abeta) production, clearance, and degradation. Neprilysin has recently been implicated as a major extracellular Abeta degrading enzyme in the brain. However, there has been no direct demonstration that neprilysin antagonizes the deposition of amyloid-beta in vivo. To address this issue, a lentiviral vector expressing human neprilysin (Lenti-Nep) was tested in transgenic mouse models of amyloidosis. We show that unilateral intracerebral injection of Lenti-Nep reduced amyloid-beta deposits by half relative to the untreated side. Furthermore, Lenti-Nep ameliorated neurodegenerative alterations in the frontal cortex and hippocampus of these transgenic mice. These data further support a role for neprilysin in regulating cerebral amyloid deposition and suggest that gene transfer approaches might have potential for the development of alternative therapies for Alzheimer's disease.

Alzheimer Disease↗
PubMed2003

Regional alterations in amyloid precursor protein and nerve growth factor across age in a mouse model of Down's syndrome.

Individuals with Down's syndrome (DS) develop the pathological hallmarks of Alzheimer's (AD) disease at an early age, subsequently followed by memory decline and dementia. We have utilized an animal model for DS, mice with segmental trisomy of chromosome 16 (Ts65Dn), to study biological events linked to memory loss. Previous studies demonstrated a cognitive decline and loss of cholinergic markers after 6-8 months of age. In the current study, we found increased levels of amyloid precursor protein (APP) in the striatum by 6-8 months of age, and in the hippocampus and parietal cortex by 13-16 months of age in Ts65Dn but not in normosomic mice. Additionally, Ts65Dn mice exhibited alterations in nerve growth factor (NGF) levels in the basal forebrain and hippocampus. Ts65Dn mice demonstrated a significant decline in NGF levels in the basal forebrain with age, as well as a reduction in hippocampal NGF by 13-16 months of age. These findings demonstrate that elevated APP and decreased NGF levels in limbic areas correlate with the progressive memory decline and cholinergic degeneration seen in middle-aged trisomic mice.

Age Factors↗
PubMed2002

Inflammation-dependent cerebral deposition of serum amyloid a protein in a mouse model of amyloidosis.

The major pathological hallmark of amyloid diseases is the presence of extracellular amyloid deposits. Serum amyloid A (SAA) is an apolipoprotein primarily produced in the liver. Serum protein levels can increase one thousandfold after inflammation. SAA is the precursor to the amyloid A protein found in deposits of systemic amyloid A amyloid (AA or reactive amyloid) in both mouse and human. To study the factors necessary for cerebral amyloid formation, we have created a transgenic mouse that expresses the amyloidogenic mouse Saa1 protein in the brain. Using the synapsin promoter to drive expression of the Saa1 gene, the brains of transgenic mice expressed both RNA and protein. Under noninflammatory conditions, transgenic mice do not develop AA amyloid deposits in the brain; however, induction of a systemic acute-phase response in transgenic mice enhanced amyloid deposition. This deposition was preceded by an increase in cytokine levels in the brain, suggesting that systemic inflammation may be a contributing factor to the development of cerebral amyloid. The nonsteroidal anti-inflammatory agent indomethacin reduced inflammation and protected against the deposition of AA amyloid in the brain. These studies indicate that inflammation plays an important role in the process of amyloid deposition, and inhibition of inflammatory cascades may attenuate amyloidogenic processes, such as Alzheimer's disease.

Age Factors↗
PubMed2002

Apolipoprotein E modulates Alzheimer's Abeta(1-42)-induced oxidative damage to synaptosomes in an allele-specific manner.

Several functional differences have been reported among the three human e2, e3, and e4 alleles of apolipoprotein E (apoE). One functional difference lies in the antioxidant potential of these alleles; e4 has the poorest potential. Interestingly, e4 also correlates with increased oxidative damage in the Alzheimer's disease (AD) brain, which may explain why the inheritance of the e4 allele is a risk factor for the onset of AD. Beta-amyloid (Abeta) is also intimately involved in AD and promotes oxidative damage in vitro; therefore, we have examined the role of the different apoE alleles in modulating Abeta(1-42)-induced oxidation to synaptosomes. Measurement of specific markers of oxidation in synaptosomes isolated from mice that express one of the human apoE alleles indicates that Abeta-induced increases of these markers can be modulated by apoE in an allele-dependent manner (e2>e3>e4). Increases in reactive oxygen species formation and protein and lipid oxidation were always greatest in e4 synaptosomes as compared to e2 and e3 synaptosomes. Our data support the role of apoE as a modulator of Abeta toxicity and, consistent with the antioxidant potentials of the three alleles, suggest that the e4 allele may not be as effective in this role as the e2 or e3 alleles of apoE. These results are discussed with reference to mechanistic implications for neurodegeneration in the AD brain.

Aldehydes↗
PubMed2002

Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I, but not apoB, in human apoB transgenic mice.

Scavenger receptor BI (SR-BI) is a multi-ligand lipoprotein receptor that mediates selective lipid uptake from HDL, and plays a central role in hepatic HDL metabolism. In this report, we investigated the extent to which SR-BI selective lipid uptake contributes to LDL metabolism. As has been reported for human LDL, mouse SR-BI expressed in transfected cells mediated selective lipid uptake from mouse LDL. However, LDL-cholesteryl oleoyl ester (CE) transfer relative to LDL-CE bound to the cell surface (fractional transfer) was approximately 18-fold lower compared with HDL-CE. Adenoviral vector-mediated SR-BI overexpression in livers of human apoB transgenic mice ( approximately 10-fold increased expression) reduced plasma HDL-cholesterol (HDL-C) and apolipoprotein (apo)A-I concentrations to nearly undetectable levels 3 days after adenovirus infusion. Increased hepatic SR-BI expression resulted in only a modest depletion in LDL-C that was restricted to large LDL particles, and no change in steady-state concentrations of human apoB. Kinetic studies showed a 19% increase in the clearance rate of LDL-CE in mice with increased SR-BI expression, but no change in LDL apolipoprotein clearance. Quantification of hepatic uptake of LDL-CE and LDL-apolipoprotein showed selective uptake of LDL-CE in livers of human apo B transgenic mice. However, such uptake was not significantly increased in mice over-expressing SR-BI. We conclude that SR-BI-mediated selective uptake from LDL plays a minor role in LDL metabolism in vivo.

Adenoviridae↗
PubMed2002

The fate of HDL particles in vivo after SR-BI-mediated selective lipid uptake.

Scavenger receptor class B type I (SR-BI) delivers cholesterol ester from HDL to cells via a selective uptake mechanism, whereby lipid is transferred from the core of the particle without concomitant degradation of the protein moiety. The precise metabolic fate of HDL particles after selective lipid uptake is not known. To characterize SR-BI-mediated HDL processing in vivo, we expressed high levels of this receptor in livers of apoA-I(-/-) mice by adenoviral vector gene transfer, and then injected the mice with a bolus of human HDL(2) traced with (125)I-dilactitol tyramine. HDL recovered from apoA-I(-/-) mice over-expressing SR-BI was significantly smaller than HDL recovered from control mice as measured by non-denaturing gel electrophoresis. When injected into C57BL/6 mice, these HDL "remnants" were rapidly converted to HDL(2)-sized lipoprotein particles, and were cleared from the plasma at a rate similar to HDL(2). In assays in cultured cells, HDL remnants did not stimulate ATP-binding cassette transporter A1-dependent cholesterol efflux. When mixed with mouse plasma ex vivo, HDL remnants rapidly converted to larger HDL particles. These studies identify a previously ill-defined pathway in HDL metabolism, whereby SR-BI generates small, dense HDL particles that are rapidly remodeled in plasma. This remodeling pathway may represent a process that is important in determining the rate of apoA-I catabolism and HDL-mediated reverse cholesterol transport.

ATP Binding Cassette Transporter 1↗